EP1299410A2 - GFR alpha1-RET SPECIFIC AGONISTS AND METHODS THEREFOR - Google Patents
GFR alpha1-RET SPECIFIC AGONISTS AND METHODS THEREFORInfo
- Publication number
- EP1299410A2 EP1299410A2 EP00991427A EP00991427A EP1299410A2 EP 1299410 A2 EP1299410 A2 EP 1299410A2 EP 00991427 A EP00991427 A EP 00991427A EP 00991427 A EP00991427 A EP 00991427A EP 1299410 A2 EP1299410 A2 EP 1299410A2
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- Prior art keywords
- seq
- ret
- growth factor
- gdnf
- cell
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- C07K2319/00—Fusion polypeptide
Definitions
- This invention relates generally to trophic or growth factors and, more particularly, to chimeric GDNF family growth factors which activate GFR ⁇ l-RET but do not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET, growth factors derived therefrom and methods therefor.
- Neurotrophic factors promote ' differentiation, maintain a mature phenotype and provide trophic support, promoting growth, function and survival of neurons.
- Nerve growth factor was the first neurotrophic factor to be identified and characterized (Levi-Montalcini et al., J. Exp . Zool . 11 6: 321, 1951).
- NGF exists as a non-covalently bound homodimer that promotes the survival and growth of sympathetic, neural crest-derived sensory, and basal forebrain cholinergic neurons.
- Other effects of NGF including effects on non-neuronal cells of the endocrine and immune systems (including inflammatory cells) are disclosed in, e.g., Levi-Montalcmi and Booker, Proc Na t ' l Acad Sci 46: 384-391, 1960; Johnson et al. Science 210 : 916-918,
- TGF- ⁇ family members have 7 canonical framework cysteme residues which identify members of this superfamily.
- the NGF family is the prototype of such a family of growth factors.
- Bram-derived neurotrophic factor the second member of this family to be discovered, was shown to be related to NGF by virtue of the conservation of all six cystemes that form the three internal disulfides of the NGF monomer (Barde, Prog Growth Factor Res 2 : 237-248, 1990 and Liebrock et al . Na ture 341 : 149-152, 1989).
- additional members NT-3, NT-4/5) of this neurotrophm family were rapidly found by several groups (Klein, FASEB J 8 : 738-44, 1994).
- the known members of this subfamily of the TGF- ⁇ superfamily include glial cell line-derived neurotrophic factor (GDNF), neurtu ⁇ n, persephin, and artemm.
- GDNF glial cell line-derived neurotrophic factor
- neurtu ⁇ n glial cell line-derived neurotrophic factor
- persephin neurtunn
- persephin neurtunn
- artemm artemm
- the placement of GDNF, neurtunn, persephin, and artemm into the same growth factor family, also referred to as the GDNF ligand family is based on the similarities of their physical structures and biological activities.
- human persephin has about 40% sequence identity and about 43% sequence conservation with human GDNF; about 49% sequence identity and about 50% sequence conservation with human neurturin; and about 45% sequence identity and about 48% sequence conservation with human artemin.
- these four proteins have the seven cysteine residues typical of TGF- ⁇ family members.
- the GDNF family ligands support the survival of dopaminergic ventral midbrain neurons cultured from the embryo. Additionally, GDNF, neurturin, and persephin support the survival of spinal and facial motor neurons in both in vi tro survival and in vivo injury paradigms, identifying these ligands as potential therapeutic agents in the treatment of neurodegenerative diseases (Henderson et al., Science 266: 1062-1064, 1994; Horger et al . , J Neurosci . 18 : 4929-37, 1998; Klein et al .
- the GDNF family ligands share receptors and signal transduction pathways (Creedon et al . , Proc. Na tl . Acad. Sci . USA 94 : 7018-7023, 1997; Durbec et al., Na ture 381 : 789-793, 1996; Trupp et al . , Na ture 381 : 785-789, 1996; Baloh et al . , Neuron 18 : 793-802, 1997).
- GFR ⁇ a transmembrane signal transducing component
- RET Ret protein-tyrosine kinase
- the members of the GFR ⁇ family include GFR ⁇ l (previously known as GDNFR ⁇ , TrnRl and RetLl), GFR ⁇ 2 (previously TrnR2, NTNR ⁇ and RetL2), GFR ⁇ 3 (previously TrnR3) (GFR ⁇ Nomenclature Committee, Neuron 19 : 485, 1997) and possibly GFR ⁇ 4 , a receptor currently only identified in the chicken (cGFR ⁇ 4) (Enokido et al . , Curren t Biology 8 : 1019-1022, 1998) .
- Persephin does not bind or activate any of the known mammalian GFR ⁇ 's but does bind to chicken GFR ⁇ 4 (Milbrandt et al . , supra ; Baloh et al . , 1998, supra , Enokido et al . supra ) . However, despite these preferred interactions, there is also clear cross-talk between the different ligand-receptor pairs.
- GFR ⁇ l is the only physiologically critical GDNF receptor in kidney organogenesis and enteric nervous system development (Cacalano et al., Neuron 21 : 53-62,1998; Enomoto et al . , Neuron 21 : 317-324,1998).
- GDNF-deficient mice have greater losses in peripheral ganglia than GFR ⁇ l-deficient mice, suggesting that GDNF can utilize other receptors to support survival of peripheral neurons, likely GFR ⁇ 2-RET (Cacalano et al., supra ; Enomoto et al., supra ) .
- GFR ⁇ 2 expression is diffuse and weak in the pars compacta region of the substantia nigra, and does not colocalize with tyrosine hydroxylase staining (TH) neurons, in contrast to the significantly stronger expression of GFR ⁇ l, which does colocalize with TH staining neurons (Horger et al . , J. Neurosci . 18 : 4929-4937, 1998).
- GDNF disulfide-bonded dimer that is significantly similar to the structure of TGF- ⁇ 2, as predicted by the cysteine spacing of its primary sequence (Daopin et al., Science 257: 369-373, 1992; Eigenbrot and Gerber, Na t . Struct . Biol . 4 : 435-438, 1997; Schlungger and Grutter, Na ture 358 : 430-434, 1992) .
- GDNF neurotrophic factor
- the inventors have succeeded in discovering that certain growth factors can be constructed which activate GFR ⁇ l-RET but which do not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET.
- Reference to the term "substantially” is intended to mean that the growth factor is selective for GFR ⁇ l-RET and does not activate GFR ⁇ 2- RET or GFR ⁇ 3-RET to an extent greater than that of persephin, which is considered to be ineffective in binding to or activating GFR ⁇ 2-RET or GFR ⁇ 3-RET, and/or preferably, no more than 50%, more preferably, no more than 40%, still more preferably, no more than 30% and, most preferably, no more than 20% as effective as GDNF in activating GFR ⁇ 2-RET or artemin in activating GFR ⁇ 3-RET.
- the present invention is directed to growth factors which activate GFR ⁇ l-RET but do not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET.
- growth factors can be chimeric GDNF family ligands or derivatives thereof.
- the derivatives can be peptidyl derivatives or non-peptidyl derivatives.
- Preferred peptidyl derivatives are GDNF family growth factors having conservative amino acid substitutions.
- the growth factor can be based upon persephin which does not act upon GFR ⁇ l-RET.
- the chimeric growth factor thus, preferably comprises a substituted persephin having different amino acids in the F2a and/or F2c regions than those in a naturally occurring persephin.
- F2 represents the finger 2 stretch of amino acids as determined for GDNF (Eigenbrot and Gerber, 1997, supra ) and the F2a and F2c regions are portions of finger 2 as shown in Figure 1.
- the F2a region of the substituted persephin preferably comprises from one to eight amino acids identical to region F2a of GDNF, neurturin or artemin, i.e. from one to eight amino acids can be the same and in the same position in the F2a region of the substituted persephin as in the F2a region of GDNF, neurturin or artemin.
- the amino acid in a particular position in the F2a region of the substituted persephin can be a conservative amino acid substitution for the amino acid in that position in the F2a region of GDNF, neurturin or artemin.
- Persephin lacks an amino acid between position 71 and 72 which is present in the aligned F2a regions of GDNF and neurturin and, hence, reference to amino acid substitutions in the F2a region of persephin is intended to include an insertion between positions 71 and 72 with an amino acid in that aligned position from GDNF, neurturin or artemin or a conservative amino acid substitution therefor.
- the F2c region of the substituted persephin of this embodiment preferably comprises from one to eight amino acids identical to region F2c of GDNF, neurturin or artemin, i.e. from one to eight amino acids can be the same and in the same position in the F2c region of the substituted persephin as in the F2c region of GDNF, neurturin or artemin.
- the amino acid in a particular position in the F2c region of the substituted persephin can be a conservative amino acid substitution for the amino acid in that position in the F2c region of GDNF, neurturin or artemin.
- the growth factor of the present invention comprises a substituted human persephin (SEQ ID NO:l), mouse persephin (SEQ ID NO : 2 ) or rat persephin (SEQ ID NO: 3) having substitutions in the F2a and/or F2c regions.
- the persephin sequence can also include variations of these persephin sequences, having conservative amino acid substitutions outside of the F2a and F2c regions.
- the F2a and F2c regions in the substituted persephin sequences have amino acids substitutions from the F2a and F2c regions of human GDNF (SEQ ID NO: 4), mouse GDNF (SEQ ID NO: 5), rat GDNF (SEQ ID NO: 6), human neurturin (SEQ ID NO : 7 ) , mouse neurturin (SEQ ID NO: 8), human artemin (SEQ ID NO: 9), mouse artemin (SEQ ID NO: 10).
- the growth factor of the present invention comprises a substituted human persephin having F2a and F2c region substitutions independently selected from corresponding regions of human GDNF, human neurturin or human artemin.
- the growth factor comprises SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25; the most preferred growth factor consists of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO:28.
- the present invention also includes compositions comprising a pharmaceutically acceptable preparation of the GFR ⁇ l-RET selective growth factors of the present invention.
- the present invention is directed to a nucleic acid comprising a polynucleotide encoding a growth factor which activates GFR ⁇ l-RET but does not substantially activate GFR ⁇ 2- RET or GFR ⁇ 3-RET.
- the encoded growth factor is preferably a chimeric GDNF family ligand or a conservatively substituted derivative thereof.
- the encoded chimeric growth factor preferably, comprises a persephin having the F2a and/or F2c region substituted with from one to eight amino acids identical to and in the same position as the amino acid in the F2a region or F2c region, respectively, of GDNF, neurturin or artemin.
- the encoded F2a region or F2c region amino acid substitutions can comprise a conservative amino acid substitution for the amino acid in that position in the F2a region or F2c region of GDNF, neurturin or artemin.
- the encoded, substituted persephin is a substituted human persephin (SEQ ID NO:l), mouse persephin (SEQ ID NO : 2 ) or rat persephin (SEQ ID NO: 3) having amino acid substitutions in the F2a and/or F2c regions.
- the encoded persephin sequence can also include variations of these persephin sequences, having conservative amino acid substitutions outside of the F2a and F2c regions.
- the F2a and F2c regions in the encoded chimeric persephin sequences have amino acid substitutions from the F2a and F2c regions of human GDNF (SEQ ID NO : 4 ) , mouse GDNF (SEQ ID NO: 5), rat GDNF (SEQ ID NO: 6), human neurturin (SEQ ID NO: 7), mouse neurturin (SEQ ID NO: 8), human artemin (SEQ ID NO: 9), or mouse artemin (SEQ ID NO: 10).
- the encoded sequence comprises a substituted human persephin having F2a and F2c region substitutions independently selected from corresponding sequences of human GDNF, human neurturin or human artemin. More preferably, the encoded sequence comprises SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25; the most preferred encoded growth factor consists of SEQ ID NO: 26, SEQ ID NO:27 or SEQ ID NO:28.
- the present invention comprises a vector which comprises expression regulatory elements operably linked to a polynucleotide which encodes a growth factor which activates GFR ⁇ l- RET but does not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET and which is, preferably, a chimeric GDNF family ligand.
- the present invention also encompasses a host cell which is transformed with the vector .
- the present invention provides chimeric GDNF family ligands with an altered N-terminus .
- the N- terminus can be eliminated, truncated, or substituted with the N- terminus of another GDNF family member or another protein such as a member of another TGF- ⁇ superfamily member.
- These chimeras retain the GFR ⁇ -RET activating ability of the GDNF family ligand.
- the growth factors of the present invention can be used to provide trophic support or produce differentiation in a mammalian cell.
- the present invention comprises a method for providing trophic support to a mammalian cell or for producing differentiation of a mammalian cell or both.
- the method comprises treating the cell with an effective amount of a growth factor which activates GFR ⁇ l-RET but does not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET.
- the growth factor is preferably a chimeric GDNF family ligand or a conservatively substituted derivative thereof.
- the chimeric growth factor preferably, comprises a persephin having the F2a region and/or F2c region substituted with from one to eight amino acids identical to and in the same position as that amino acid in the F2a region or F2c region, respectively, of GDNF, neurturin or artemin.
- the F2a region or F2c region amino acid substitutions can comprise a conservative amino r acid substitution for the amino acid in that position in the F2a region or F2c region of a GDNF, neurturin or artemin.
- the substituted persephin is a substituted human persephin (SEQ ID NO:l), mouse persephin (SEQ ID N0:2) or rat persephin (SEQ ID NO:3) having amino acid substitutions in the F2a and/or F2c regions.
- the persephin sequence can also include variations of these persephin sequences, having conservative amino acid substitutions outside of the F2a and F2c regions.
- the F2a and F2c regions in the substituted or chimeric persephin sequences preferably have amino acid substitutions from the F2a and F2c regions of human GDNF (SEQ ID NO: ), mouse GDNF (SEQ ID NO: 5), rat GDNF (SEQ ID NO: 6), human neurturin (SEQ ID NO: 7), mouse neurturin (SEQ ID NO: 8), human artemin (SEQ ID NO: 9), or mouse artemin (SEQ ID NO: 10).
- the growth factor of the present invention comprises a substituted human persephin having F2a and F2c region substitutions independently selected from corresponding regions of human GDNF, human neurturin or human artemin.
- the growth factor comprises SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25; the most preferred growth factor consists of SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.
- the mammalian cell can be any cell which contains GFR ⁇ l-RET, preferably, a neuronal cell, a he atopoietic cell or a cardiac muscle cell.
- the treatment can further comprise administering to the cell a GFR ⁇ l polypeptide.
- the cell can be treated with a nucleic acid comprising a polynucleotide which encodes for expression, a growth factor which activates GFR ⁇ l-RET but does not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET.
- the encoded growth factor is preferably a chimeric GDNF family ligand or a conservatively substituted derivative thereof.
- the cell can also be treated with a GFR ⁇ l polypeptide or a polynucleotide encoding a GFR ⁇ l polypeptide.
- the cell can be in an patient and the treatment comprises administering the growth factor to the patient.
- the method also encompasses administering a polynucleotide encoding the growth factor to the patient or implanting into the patient a cell which expresses the growth factor.
- the cell can be a neuronal cell in a patient suffering from peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, diabetes, AIDS, ischemic stroke, acute brain injury, acute spinal cord injury, a nervous system tumor such as neuroblastoma, multiple sclerosis, infection, side effects of chemotherapy, or an enteric disease such as idiopathic constipation or constipation associated with Parkinson's disease, spinal cord injury or use of opiate pain-killers.
- the cell can also be a non- neuronal cell such as, for example, in a patient suffering from small cell lung carcinoma.
- the cell can also be a hematopoietic cell in a patient suffering from eosinopenia, basopenia, lymphopenia, monocytopenia, neutropenia, anemia, thrombocytopenia, or stem-cell insufficiency or a cardiac muscle cell in a patient suffering from cardiomyopathy or congestive heart failure.
- the present invention comprises a method for preventing or treating cellular degeneration or insufficiency in an individual. The method comprises administering to the individual a therapeutically effective amount of a growth factor which activates GFR ⁇ l-RET but does not substantially activate GFR ⁇ 2-RET or GFR ⁇ 3-RET.
- the growth factor is preferably, a chimeric GDNF family ligand or a conservatively substituted derivative thereof.
- the chimeric growth factor preferably, comprises a persephin having the F2a region and/or F2c regions independently substituted with from one to eight amino acids identical to and in the same position as that amino acid in the F2a region or F2c region, respectively, of GDNF, neurturin or artemin.
- the F2a region or F2c region amino acid substitutions can comprise a conservative amino acid substitution for the amino acid in that position in the F2a region or F2c region of
- the substituted persephin is a substituted human persephin (SEQ ID NO:l), mouse persephin (SEQ ID NO:2) or rat persephin (SEQ ID NO:3) having amino acid substitutions in the F2a and/or F2c regions.
- the persephin sequence can also include variations of these persephin sequences, having conservative amino acid substitutions outside of the F2a and F2c regions.
- the F2a and F2c regions in the substituted or chimeric persephin sequences preferably have amino acid substitutions from the F2a and F2c regions of human GDNF (SEQ ID NO: 4), mouse GDNF (SEQ ID NO: 5), rat GDNF (SEQ ID NO:6), human neurturin (SEQ ID NO:7), mouse neurturin (SEQ ID NO: 4), mouse GDNF (SEQ ID NO: 5), rat GDNF (SEQ ID NO:6), human neurturin (SEQ ID NO:7), mouse neurturin (SEQ ID NO:
- the growth factor comprises a substituted human persephin having F2a and F2c region substitutions independently selected from corresponding regions of human GDNF, human neurturin or human artemin. More preferably, the growth factor comprises SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25; the most preferred growth factor consists of SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.
- the mammalian cell can be any cell which contains GFR ⁇ l-RET, preferably, a neuronal cell, a hematopoietic cell or a cardiac muscle cell.
- the treatment can further comprise administering to the individual a GFR ⁇ l polypeptide.
- a polynucleotide encoding the growth factor can be administered to the individual.
- a cell which expresses the growth factor can be implanted into the individual.
- the treatment with the growth factor can be facilitated by also administering to the individual a GFR ⁇ l polypeptide or a polynucleotide encoding a GFR ⁇ l polypeptide or a cell which expresses a GFR ⁇ l polypeptide can be implanted into the individual .
- the growth factor or polynucleotide encoding the growth factor can be administered to an individual suffering from (a) neuronal degeneration resulting from peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, diabetes, AIDS, ischemic stroke, acute brain injury, acute spinal cord injury, a nervous system tumor such as neuroblastomas, multiple sclerosis, infection, chemotherapy side effects, or an enteric disease such as idiopathic constipation or constipation associated with Parkinson's disease, spinal cord injury or use of opiate pain-killers, (b) from small cell lung carcinoma,
- mimics of neurturin are provided which activate GFR ⁇ l-RET and GFR ⁇ 2-RET but do not substantially activate GFR ⁇ 3-RET.
- These mimics comprise persephin having amino acid substitutions in regions Ha, F2a and F2c from neurturin, or conservative amino acid substitutions therefor.
- Nucleic acids comprising polynucleotides encoding these mimics are also provided. These mimics can substitute for neurturin, for example in methods for providing trophic support to a mammalian cell or producing differentiation of a mammalian cell, or in methods for preventing or treating cellular degeneration or insufficiency in an individual.
- mimics of artemin are also provided which activate GFR ⁇ l- RET and GFR ⁇ 3-RET but do not substantially activate GFR ⁇ 2-RET.
- These mimics comprise persephin having amino acid substitutions in regions Ha, F2a and F2c from artemin, or conservative amino acid substitutions therefor.
- Nucleic acids comprising polynucleotides encoding these mimics are also provided. These mimics can substitute for artemin, for example in methods for providing trophic support to a mammalian cell or producing differentiation of a mammalian cell, or in methods for preventing' or treating cellular degeneration or insufficiency in an individual.
- chimeric GDNF family ligands which can be used to prevent the atrophy, degeneration or death of certain cells, in particular, neurons in need of trophic support; the provision of polynucleotides encoding chimeric GDNF family ligands for use in gene therapy; the provision of methods for obtaining chimeric GDNF family ligands by recombinant techniques; the provision of methods for providing trophic support to target cells, particularly neurons; and the provision of methods for treating disease conditions involving cellular degeneration, and in particular, neuronal dysfunction and degeneration.
- Figure 1 illustrates the amino acid sequence alignment of rat GDNF (comprising SEQ ID NO: 6); mouse neurturin (comprising SEQ ID NO: 7);
- mice artemin comprising SEQ ID NO: 10
- mouse persephin comprising SEQ ID NO:2
- sequences include the region N- terminal to the first canonical cysteine and C-terminal to the seventh canonical cysteine, where the canonical cysteines are indicated by a thick outline, and where secondary structure elements (' ⁇ ' for ⁇ -helix, ' ⁇ ' for ⁇ -strand and '3' for 3 ⁇ 0 -helix) as determined for GDNF (Eigenbrot and Gerber, Na t . Struct . Biol .
- FIG. 2A depicts a schematic diagram of wild-type and tagged GDNF constructs, where the site of proprotein convertase cleavage of GDNF is indicated by the arrow (SS - signal sequence, PRO - pro domain, MATURE - mature processed monomer) , where GDNF with a 6- histidine and FLAG ® tag inserted two residues after the RXXR cleavage site is designated "G-hf-GDNF" and where GDNF with the histidine and FLAG ® tag wherein the GDNF pro sequence is substituted with the neurturin pro sequence is designated "N-hf-GDNF”;
- Figure 2B is an image of a FLAG ® immunoblot demonstrating the production of tagged GDNF proteins in COS cells, where the immunoblot indicates that N-hf-GDNF produces GDNF with the 40 amino acid N- ter inal extension replaced by two amino acids and the tandem His- FLAG tags;
- Figure 2C is a graph showing the ability of
- Figure 3B is a photograph of an anti-FLAG ® immunoblot of homologue scanning mutants from transfected COS cell lysates, where the two major bands indicated by arrows represent the unprocessed (top) and mature (bottom) forms of the mutants, since the lower band corresponds in size to the secreted product as shown in Figure 2B;
- Figure 4A is a graph showing relative GFR ⁇ l-RET receptor activation by homologue-scanning GDNF mutants, where values are represented as a percentage of reporter activation by GDNF and the mean and standard deviation of triplicate measurements from a representative experiment are shown, along with arbitrary boundary lines at 33% and 66% of wild-type GDNF activation;
- Figure 4B depicts a space-filling model of the GDNF crystal structure, where the upper representation is a side-view of the GDNF dimer, and the lower representation is from a viewpoint below the dimer, showing putative critical GFR ⁇ l-RET interaction domains Hb, F2a, and F2c
- Figure 4D depicts a space-filling model of the GDNF crystal structure, where the upper representation is a side-view of the GDNF dimer, and the lower representation is from a viewpoint below the dimer, showing putative critical GFR ⁇ 2-RET interaction domains Ha, F2a, and F2c;
- Figure 5A is a schematic representation of G-hf-GNDF, P-hf- PSPN, and GDNF/persephin replacement chimeras (PGP) for gain-of- function analysis, where the persephin amino acid sequence in parentheses below the schematics was replaced by the GDNF sequence shown above;
- Figure 5B is a graph showing the ability of PGP-F2ac to activate the GFR ⁇ l-RET receptor and the inability of other PGP chimeras and P-hf-PSPN to activate GFR ⁇ l-RET;
- Figure 5C is an image of a RET immunoblot of anti-FLAG ® - immunoprecipitated lysate from neuro2a cells either untransfected (RET) or transfected with FLAG ® -tagged GFR ⁇ l (RET + GFR ⁇ l) treated with the indicated factor at 25 ng/mL, where a fraction of the total lysate shows a RET doublet, with the upper band corresponding to the mature cell-surface form;
- Figure 5D is a histogram of BrdU incorporation by NBL-S neuroblastoma cells in the presence of the indicated factors purified from conditioned medium of transiently transfected COS cells, showing that mutant PGP-F2ac stimulates proliferation of NBL-S cells at a similar level to GDNF at all doses tested;
- Figure 5E are photographs of SH-SY5Y neuroblastoma cells cultured in the presence of no factor (CTRL) , retinoic acid (RA; lO ⁇ M) or conditioned medium from COS cells expressing the indicated construct, showing that GDNF and PGP-F2ac stimulates neurite outgrowth in these cells, whereas persephin does not;
- CTRL no factor
- RA retinoic acid
- Figure 6A is a schematic diagram depicting the structures of N- hf-GDNF, P-hf-PSPN, PGP-F2ac, PNP-F2ac, and PAP-F2ac;
- Figure 6B is two graphs showing the ability or inability of the five molecules depicted in Figure 6A to activate GFR ⁇ l-RET (left side) and GFR ⁇ 2-RET (right side);
- Figure 7A is a schematic diagram depicting the structures of G- hf-GDNF, P-hf-PSPN, PGP-F2ac, PNP-F2ac, PNP-Ha/F2ac, and PNP-Ha;
- Figure 7B is a graph showing the ability of inability of the six molecules depicted in Figure 7A to activate GFR ⁇ 2-RET;
- Figure 7C is a schematic diagram depicting the structures of N- hf-GDNF, N-hf-artemin, PAP-F2ac, PAP-Ha/F2ac, and P-hf-PSPN;
- Figure 7D is a graph showing the ability of inability of the five molecules depicted in Figure 7C to activate GFR ⁇ 3-RET
- Figure 8A is a graph showing survival of cerebellar granule cells transfected with the indicated receptor components and cultured for 3 days in the presence of high-potassium plus serum (K25+S), low potassium (K5), or low potassium plus GDNF (GDNF);
- Figure 8B is a graph showing survival of cerebellar granule cells transfected with GFR ⁇ l-RET or GFR ⁇ 2-RET cultured in the presence of the indicated factors .
- growth factors are, preferably, chimeric GDNF family ligand growth factors which have the ability to activate a GFR ⁇ l-RET complex, and are thus useful for promoting the survival of peripheral and central neuronal populations in vivo or in vi tro .
- the present invention provides novel chimeras which vary from the previously known naturally occurring GDNF ligand family members GDNF, artemin, neurturin, and persephin by their ability to activate mammalian GFR ⁇ l-RET without substantially activating GFR ⁇ 2-RET or GFR ⁇ 3-RET complexes.
- PGP-F2ac e.g., SEQ ID NOS: 12, 23, and 26
- PNP-F2ac e.g., SEQ ID NOS:14, 24, and 27
- PAP-F2ac e.g., SEQ ID NOS:16, 25, and 28
- the sequences are identical to human persephin (e.g., SEQ ID NO:l), mouse persephin (SEQ ID NO:2), or rat persephin (SEQ ID NO: 3) except that amino acids 63-66 in the F2a region and/or amino acids 76-82 in the F2c region (corresponding to amino acids 68- 71 and 81-87 of full length persephin, as depicted in Figure 1) are replaced with the corresponding amino acids from a GDNF in PGP-F2ac, a neurturin in PNP-F2ac, or an artemin in PAP-F2ac.
- persephin sequences do not bind to or activate GFR ⁇ l-RET, GFR ⁇ 2-RET or GFR ⁇ 3-RET
- the replacement of these amino acids of persephin with amino acids in the same aligned positions from GDNF, neurturin, or artemin confers upon the chimeric molecule the ability to bind to and activate GFR ⁇ l-RET.
- the persephin amino acids 63-66 and 76-82 are substituted with regions from growth factors which can also activate GFR ⁇ 2-RET (neurturin and GDNF) and GFR ⁇ 3-RET (artemin)
- the chimeras do not activate those receptors.
- the ability to substantially activate a GFR ⁇ - RET complex by a GDNF family ligand is defined as the ability of that ligand to activate the MAP kinase pathway at a level preferably at least about 40%, more preferably at least about 50%, and most preferably at least about 80% of the ability of GDNF to activate that pathway through GFR ⁇ l-RET activation.
- This can be measured, e.g., by measuring luciferase activity after activating RET-expressing cells transformed with expression plasmids encoding the individual GFR ⁇ receptor together with the Gal4-Elk/Gal4-Luc reporter system as previously described with GDNF (Worby et al . , J Biol Chem 273 : 3502- 3508, 1998; Worby et al . , J. Biol . Chem . 72:23619-23622, 1996).
- regions in other GDNF family ligands which are "corresponding" or “analogous” to persephin amino acids 65-71 (also known as the F2a region - Figure 1) and 81-87 (the F2c region) , starting at the first canonical cysteine, are meant to encompass the regions of those growth factors which are aligned with those persephin regions by commonly used alignment methods which are designed to align regions according to similar predicted tertiary structures.
- An example is the Clustal method (Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, WI) .
- multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments.
- Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval. Penalties for opening and lengthening gaps in the alignment contribute to the score.
- the residue weight table used for that alignment program is PAM250
- the F2a and F2c regions consist of amino acids 60-66 and 76-83 of persephin, starting at the first canonical cysteine residue (as set forth in SEQ ID NOS:l-3). When numbered from the N-terminus of persephin, as depicted in Figure 1, these regions correspond to amino acids 65-71 and 81-88, respectively.
- the F2a and F2c regions correspond to amino acids 63-71 and 80- 87, respectively, of GDNF as set forth in SEQ ID NOS: 4-6 and amino acids 103-110 and 120-127, respectively, of full length GDNF ( Figure 1), and have the sequences RPIAFDDD and YHILRKHS, respectively, in human GDNF (SEQ ID NO:4).
- F2a and F2c are amino acids 64-71 and 81-88, respectively, in SEQ ID NOS : 7 and 8, and amino acids 69-76 and 86-93, respectively, of full length neurturin ( Figure 1), and have the sequences RPTAYEDE and YHTVHELS in human neurturin (SEQ ID NO:7).
- F2a and F2c are amino acids 67-73 and 83-90, respectively, in SEQ ID NOS : 9 and 10, and amino acids 82-88 and 98- 105, respectively, of full length artemin (Figure 1), and have the sequences RPTRYEA and WRTVDRLS in human artemin (SEQ ID NO: 9).
- the seven amino acids 60-66 of the persephin F2a region correspond to the eight amino acids 63-70 of GDNF or 64-71 of neurturin because Clustal analysis creates a gap between amino acid 66 and 67 in persephin when aligned with GDNF or neurturin (see, e.g., Figure 1).
- the eighth amino acid in this region from GDNF or neurturin is not present in persephin (and artemin) .
- the chimeras need not comprise the exact amino acid sequence of the GDNF family ligand from which they were derived in order to retain the ability to activate GFR ⁇ l-RET. Rather, conservative substitutions in the chimeras are within the scope of the present invention. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains.
- one grouping of amino acids includes those amino acids have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M) ; another grouping is those amino acids having neutral and polar side chains (G, S, T, Y, C, N, and Q) ; another grouping is those amino acids having basic side chains (K, R, and H) ; another grouping is those amino acids having acidic side chains (D and E) ; another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I) ; another grouping is those amino acids having aliphatic- hydroxyl side chains (S and T) ; another grouping is those amino acids having amine-containing side chains (N, Q, K, R, and H) ; another grouping is those amino acids having aromatic side chains (F, Y, and W) ; and another grouping is those amino acids having sulfur- containing side chains (C and M) .
- Preferred conservative amino acid substitutions groups are: R-K; E-
- the chimeras of the present invention can also include modifications of the sequences identified herein, including sequences in which one or more amino acids have been inserted, deleted or replaced with a different amino acid or a modified or unusual amino acid, as well as modifications such as glycosylation or phosphorylation of one or more amino acids so long as the chimera containing the modified sequence retains the ability to activate GFR ⁇ l-RET.
- Amino acid(s) can be added to or removed from the N- terminus, C-terminus or within the amino acid sequence, provided the structural Cys residues are retained, which are believed to be required for GFR ⁇ -RET activating activity.
- SEQ ID NOS: 1-10 which are sequences only between the first and seventh canonical cysteines of the GDNF family members, are believed to be the minimum length required for activity of these growth factors.
- substitution of persephin amino acids with amino acids from a GFR ⁇ l-RET activating growth factor need not encompass all of amino acids 60-66 and 76-83 (numbering of SEQ ID NOS: 1-3).
- the GFR ⁇ l-RET activating chimeras PGP-F2ac, PNP-F2ac, and PAP-F2ac all comprise only the substitution of F2a amino acid residues 63-66 and F2c amino acid residues 76-82 (residue 83 is an S in all known GDNF family members) .
- GDNF, neurturin, and artemin amino acids in these regions reveals that the only GDNF, neurturin, and artemin amino acids which do not also occur in persephin are those at position 65, 77, 78, 80, and 81, as well as the amino acid which is "missing" between amino acid 66 and 67 in persephin.
- the "missing" amino acid between 66 and 67 is a D m known GDNF sequences and an E in known neurturin, which are both acidic ammo acids. However, this ammo acid is also missing m artemin which activates GFR ⁇ l.
- the provision of that missing amino acid in a persephin chimera is unlikely to influence the ability of the chimera to activate GFR ⁇ l.
- the known persephms have an A or a T, which are both uncharged at physiological pH, whereas the GFR ⁇ l-activatmg growth factors have an E or a D, which are both acidic.
- the persephms have a Q, which is uncharged and polar, whereas known GFR ⁇ l-RET activating growth factors have an R or an H, which are basic.
- the persephms have a Q (uncharged and polar) or an R (basic)
- the GFR ⁇ l-RET activating growth factors have a T (uncharged and polar) or an I (nonpolar)
- the persephms have P (nonpolar)
- the GFR ⁇ l-RET activating growth factors have D, H, (both acidic) , R (basic) , and Q (uncharged and polar) .
- the persephms have a Q (uncharged and polar)
- the GFR ⁇ l-RET activating growth factors have an H (basic) , an E (acidic), or a K (basic) .
- the substitutions at residues 65 and 77 represent an unequivocal change to a particular charge or polarity from the "nonfunctional" (i.e., does not activate GFR ⁇ l-RET) persephin to the "functional" (GFR ⁇ l-RET activating) GDNF family ligands. Therefore, these "hot spots" may be particularly important for GFR ⁇ l-RET activation.
- GFR ⁇ l-RET activation can be achieved with a persephin wherein (a) from 1 to 4 of ammo acids 63-66 are substituted with the corresponding ammo acids of a GFR ⁇ l-RET activating GDNF family ligand, or a conservatively substituted variant thereof, including at least the substitution of ammo acid 65 with an acidic ammo acid; and (b) from 1 to 7 of ammo acids 76-82 are substituted with the corresponding amino acids of a GFR ⁇ l-RET activating GDNF family ligand, or a conservatively substituted variant thereof, including the substitution of ammo acid 77 with a basic amino acid.
- Preferred substitutions at position 65 are E and D.
- Preferred substitutions at position 77 are K, R, or H; most preferred are R and H.
- the present invention provides chimeric
- GDNF family ligands comprising a persephin wherein all three of the F2a, the F2c, and the Ha regions are substituted with the equivalent regions of a GFR ⁇ -RET activating GDNF family ligand.
- the Ha region encompasses ammo acids 33-41 of persephin SEQ ID NOS: 1-3 (equivalent to amino acids 38-46 of full length persephin - Figure 1) and equivalent regions (as determined by Clustal analysis) of other GDNF family ligands (figure 1).
- the addition of the Ha region provides the ability to activate GFR ⁇ 2-RET or GFR ⁇ 3-RET, depending on the source of the Ha region.
- the Ha region (along with F2a and F2c) is derived from neurturin (e.g., PNP-Ha/F2ac - figure 7A)
- the chimera will activate GFR ⁇ 2-RET
- the Ha region (along with F2a and F2c) is derived from artemin (e.g., PAP-Ha/F2ac - figure 7C) the chimera will activate GFR ⁇ 3-RET.
- GFR ⁇ 2-RET or GFR ⁇ 3-RET activation of GFR ⁇ 2-RET or GFR ⁇ 3-RET by these chimeras is generally about 70% of the activation of that GFR ⁇ -RET by the natural (e.g., neurturin or artemin) ligand (see, e.g., figure 7). Because neurturin does not substantially bind to or activate GFR ⁇ 3, chimeras such as PNP-Ha/F2ac would be expected to activate only GFR ⁇ l-RET and GFR ⁇ 2-RET and not GFR ⁇ 3-RET or other GFR ⁇ -RET complexes having similar additional binding and activation requirements.
- chimeras such as PAP-Ha/2ac would be expected to activate only GFR ⁇ l- RET and GFR ⁇ 3-RET and not GFR ⁇ 2-RET or other GFR ⁇ -RET complexes having similar additional binding and activation requirements.
- PNP-Ha/F2ac and PAP-Ha/F2ac thus represent mimics of neurturin and artemin, respectively.
- a mimic of a GDNF family ligand is a chimera which activates the same GFR ⁇ -RET receptor as the GDNF family ligand but which primarily comprises a different GDNF family ligand. Because mimics such as PNP-Ha/F2ac and PAP-Ha/F2ac activate the same GFR ⁇ -RET receptors as neurturin and artemin, respectively, it is believed that they can substitute for those GDNF family ligands in any treatment regime, such as described in U.S. Patent No. 5,843,914 for neurturin, or U.S. Patent Application No. 09/218,698 for artemin, both of which are hereby incorporated by reference .
- the persephin chimeras with the F2a, F2c, and Ha chimeric regions can encompass conservatively substituted and modified amino acids.
- the chimeric regions from the GFR ⁇ l-RET activating ligands can also comprise only partial substitutions, as previously discussed.
- the present invention provides chimeric GDNF family ligands with an altered N-terminal region. It has been discovered that the region N-terminal to the first cysteine residue is not necessary to activate a GFR ⁇ /RET (see Example 1) . Thus, any GDNF family ligand with an N-terminal region from any other GDNF family ligand, or with any other sequence of about 100 amino acids or less (including a lack of an N-terminal region) would be expected to activate the GFR ⁇ -RET which the wild-type ligand activates. Thus, this embodiment comprises a GDNF family member with from zero to 100 amino acids N-terminal to the first cysteine residue.
- This N-terminal region can comprise a functional sequence, such as a signal sequence or a prepro region comprising a signal sequence. It is well known in the art that many proteins are synthesized within a cell with a signal sequence at the N-terminus of the mature protein sequence and the protein carrying such a leader sequence is referred to as a preprotein.
- the pre region can serve as a signal peptide which directs transport of the protein to subcellular organelles or outside of the cell.
- the pre- portion of the protein is cleaved during cellular processing of the protein.
- many proteins contain a distinct pro sequence that describes a region on a protein that is a stable precursor ⁇ f the mature protein.
- the inventors believe that the form of the GDNF family ligands which are synthesized within a cell is the pre-pro-ligand.
- the GDNF family ligand pre- region is thus generally followed by a pro- domain which is believed to preferably terminate with an RXXR consensus site immediately before the N-terminal amino acid of the mature ligand.
- Proteins synthesized with both pre- and pro- regions are referred to as preproproteins .
- GDNF family ligands containing a pre-pro- region from another GDNF family ligand or another protein, as well as polynucleotides encoding such polypeptides.
- the polypeptides can generate a mature ligand upon cleavage of the pre-pro- region and the polynucleotides can be used in an expression system to produce the polypeptide which upon cleavage of the non-artemin pre-pro- region yields mature artemin.
- non-GDNF family ligand pre-pro- region polypeptides and encoding polynucleotides are well known in the art.
- chimeras of the GDNF family ligand can comprise an N- terminal region from any protein, pro-protein, or pre-pro-protein. This can be utilized, for example, to target the protein to a particular cellular region, or outside of the cell.
- GFR ⁇ -RET activating, truncated GDNF family members (including chimeras) containing only a portion of, or entirely lacking an N- terminal region can be made.
- the chimeric GDNF family ligands identified herein may exist as dimers in their biologically active form in a manner consistent with what is known for other factors of the TGF- ⁇ superfamily.
- the chimeras may be used in homodimers or heterodimers . It is believed that monomers of the chimeras will associate under physiological conditions into homodimers.
- a preferred chimera according to the present invention is prepared in pure form by recombinant DNA technology.
- pure form or “purified form” or “substantially purified form” it is meant that a chimera composition is substantially free of other proteins which are not the chimera.
- a substantially purified chimera composition comprises at least about 50 percent chimera on a molar basis compared to total proteins or other macromolecular species present. More preferably, a substantially purified chimera composition will comprise at least about 80 to about 90 mole percent of the total protein or other macromolecular species present and still more preferably, at least about 95 mole percent or greater.
- a recombinant chimera may be made by expressing a DNA sequence encoding the chimera in a suitable transformed host cell.
- the DNA encoding the chimera may be linked to an expression vector and transformed into a host cell, and conditions established that are suitable for expression of the chimera by the transformed cell.
- Any suitable expression vector may be employed to produce a recombinant chimera such as, for example, the mammalian expression vector pCB6 (Brewer, Met ⁇ Cell Biol 43 : 233-245, 1994) or the E . coli pET expression vectors, specifically, pET-30a (Studier et al., Methods Enzymol 185 : 60-89, 1990) .
- Other suitable expression vectors for expression in mammalian and bacterial cells are known in the art as are expression vectors for use in yeast or insect cells. Baculovirus expression systems can also be employed.
- Mammalian host cells include, but are not limited to, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo 205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vi tro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells.
- monkey COS cells Chinese Hamster Ovary (CHO) cells
- human kidney 293 cells human epidermal A431 cells
- human Colo 205 cells human Colo 205 cells
- CV-1 cells other transformed primate cell lines
- normal diploid cells cell strains derived from in vi tro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells.
- Yeast strains that may act as suitable host cells include Saccharomyces cerevisiae , Schizosaccharomyces pombe, Kluyveromyces strains, Candida, and any other yeast strain capable of expressing heterologous proteins.
- Host bacterial strains include Esche ⁇ chia coli, Bacillus subtilis, Salmonella typhimurium and any other bacterial strain capable of expressing heterologous proteins. If the polypeptide is made m yeast or bacteria, it may be necessary to modify the polypeptide, for example, by phosp orylation or glycosylation of the appropriate sites using known chemical or enzymatic methods, to obtain a biologically active polypeptide.
- the chimeras of the present invention can also be expressed m transgenic plants (see, for example, U.S. Patent No. 5,679,880) or transgenic animals such as, for example, cows, goats, pigs, or sheep whose somatic or germ cells contain a nucleotide sequence encoding the chimera.
- the expressed chimera can be purified using known purification procedures, such as gel filtration and ion exchange chromatography. Purification may also include affinity chromatography using an agent that will specifically bind the chimeric polypeptide, such as a polyclonal or monoclonal antibody raised against the mature chimera or fragment thereof Other affinity resins typically used in protein purification may also be used such as concanavalm A-agarose, HEPARIN-TOYOPEARL ® or CIBACROM BLUE 3GA SEPHAROSE ® . Purification of the chimera can also include one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether.
- the chimera may be expressed as a fusion protein to facilitate purification.
- fusion proteins include the chimera am o acid sequence fused to a histidme tag, as well as the chimera ammo acid sequence fused to the ammo acid sequence of maltose binding protein (MBP) , glutathione-S- transferase (GST) or thioredox (TRX) .
- MBP maltose binding protein
- GST glutathione-S- transferase
- TRX thioredox
- the invention chimera can be tagged with a heterologous epitope, such as a FLAG ® epitope, and subsequently purified by immunoaffmity chromatography using an antibody that specifically binds the epitope. Kits for expression and purification of such fusion proteins and tagged proteins are commercially available.
- a preferred fusion protein is one containing both a histidme tag and a FLAG ® tag, e.g.
- the recombinant chimeras may also be expressed as monomeric units or such monomeric form may be produced by preparation under reducing conditions.
- refolding and renaturation can be accomplished using one of the agents noted above that is known to promote dissociation/association of proteins.
- the monomeric form can be incubated with dithiothreitol followed by incubation with oxidized glutathione disodium salt followed by incubation with a buffer containing a refolding agent such as urea.
- the chimeras of the present invention may also be produced by chemical synthesis using methods known to those skilled m the art.
- the present invention also encompasses isolated polynucleotides comprising nucleotide sequences that encode any of the chimeras described herein.
- a polynucleotide includes DNA and/or RNA and thus the nucleotide sequences recited in the Sequence Listing as DNA sequences also include the identical RNA sequences with uracil substituted for thymine residues.
- the present invention also encompasses vectors comprising an expression regulatory element operably linked to any of the chimera- encodmg nucleotide sequences included within the scope of the invention.
- This invention also includes host cells, of any variety, that have been transformed with such vectors.
- a polynucleotide which specifically hybridizes to a chimera-encodmg polynucleotide or to its complement is provided.
- Specific hybridization is defined herein as the formation of hybrids between a polynucleotide, including oligonucleotides, and a specific reference polynucleotide (e.g., a polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence encoding a chimera) wherein the polynucleotide preferentially hybridizes to the specific chimera over other GDNF family ligands.
- Specific hybridization is preferably done under high stringency conditions which, as well understood by those skilled m the art, can readily be determined by adjusting several factors during hybridization and during the washing procedure, including temperature, ionic strength, length of hybridization or washing times, and concentration of formamide (see for example, Sambrook, Fritsch and Maniatis., Molecular Cloning: a Labora tory Manual , 2d Ed. , Vols . 1 -3, Cold Spring Harbor Laboratory Press, Plainview N.Y. 11803, 1989)
- the chimeras of the present invention would be expected to promote the survival and growth of neuronal as well as non-neuronal cells (see, e.g., Examples 2 and 4 herein).
- GDNF GDNF
- neurturin neurturin
- artemin neurturin
- persephin influence a broad spectrum of neuronal populations in the peripheral and central nervous systems and the invention chimeras would be expected to also have such an effect.
- all other growth factors isolated to date have been shown to act on many different cell types (for example see Scully and Otten, Cell Biol In t 19 : 459-469, 1005; Hefti, Neurotrophic Factor Therapy 25 : 1418-1435, 1994 which are incorporated by reference) .
- NGF neurotrophic factor
- mast cells express the trk receptor and respond to NGF such that NGF is a mast cell secretogogue and survival promoting factor (Horigome et al . , J Biol Chem 269 : 2695-2707, 1994).
- members of the TGF- ⁇ superfamily act on many cell types of different function and embryologic origin. For example, GDNF activation of GFR ⁇ l is apparently critical in kidney organogenesis . Thus, it is likely that the invention chimeras will have trophic activity on a variety of different neuronal cells, both peripheral and central, as well as on non-neuronal cells.
- the present invention also includes therapeutic or pharmaceutical compositions comprising a chimera of the present invention in an effective amount for providing trophic support to cells in patients with cellular degeneration or dysfunction and a method comprising administering a therapeutically effective amount of the chimera to a cell ex vivo or in vivo .
- trophic support is used herein to mean that a growth factor such as the invention chimeras provides sufficient nourishment to a cell such that the cell maintains or recovers at least one or more of its normal functions .
- compositions and methods of the present invention are useful for treating a number of degenerative diseases and anaplastic diseases.
- the diseases include, but are not limited to peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, diabetes, AIDS, ischemic stroke, acute brain injury, acute spinal cord injury, nervous system tumors such as neuroblastomas, multiple sclerosis, peripheral nerve trauma or injury, exposure to neurotoxins, metabolic diseases such as diabetes or renal dysfunctions and damage caused by infectious agents or chemotherapy.
- compositions of the invention chimeras can be used to treat enteric diseases such as idiopathic constipation or constipation associated with Parkinson's disease, spinal cord injury or use of opiate pain-killers.
- enteric diseases such as idiopathic constipation or constipation associated with Parkinson's disease, spinal cord injury or use of opiate pain-killers.
- a chimera may be useful in treating diseases including, but not limited to disorders of insufficient blood cells such as, for example, leukopenias including eosinopenia and/or basopenia, lymphopenia, monocytopenia, neutropenia, anemias, thrombocytopenia as well as an insufficiency of stem cells for any of the above.
- the cellular degeneration or dysfunction can also involve myocardial muscle cells in diseases such as cardiomyopathy and congestive heart failure.
- small cell lung carcinoma can be treated with polypeptide or polynucleotide compositions of the invention chimeras.
- enteric diseases with the chimeras includes the treatment of enteric neuropathies.
- the enteric nervous system is a complex collection of nerves that control the function of the gastrointestinal system, including gastrointestinal motility.
- Initial clinical studies with the NT-3 have shown that this neurotrophic factor increases gastrointestinal motility in normal volunteers and in patients suffering from peripheral neuropathies. Heuckeroth et al . , Dev Biol 200 : 116-29, 1998.
- the invention chimeras as well as the other GDNF family ligands will show activity on enteric neurons.
- the chimeras will be useful in treating enteric neuropathies such as in patients suffering from severe idiopathic constipation as well as patients suffering from constipation associated with Parkinson's disease, spinal cord injury, use of opiate pain-killers, and the like.
- Whether the invention chimeras would be effective in the treatment of a particular cell type or tissues can be readily determined by one skilled in the art using any of a variety of assays known in the art.
- trophic factors can produce beneficial biochemical and morphological effects and, under some circumstances, will promote cell survival.
- neurons it is known m the art that depriving a neuron of trophic support results in a decrease m metabolic activity, i.e., glucose uptake, RNA synthesis and protein synthesis, required for normal function and growth. Deckwerth and Johnson, J. Cell Biol . 123 : 1207-1222, 1993.
- trophic support also results in a reduction in size of the cell body of the neuron. Presumably as a consequence of the loss of the metabolic effects of trophic factors, trophic factor deprivation results in a decrease or cessation of process outgrowth and may result in retraction of neuronal processes. In addition to the requirement of trophic factor for these aspects of neuronal biology, the neuron may require the neurotrophic factor to maintain survival. Thus, survival assays are a frequently used means to detect or quantitate the actions of a neurotrophic factor. However, trophic support can also be manifest as morphological, biochemical, and/or functional changes independent of neuronal number or any effect on survival.
- growth factors can produce a cell differentiation in addition to providing trophic support for cells.
- the invention chimeras and polynucleotides encoding them can be beneficially used to produce a differentiation of anaplastic cells such as cancer cells.
- the chimeras can be used to treat nervous system tumors such as neuroblastomas.
- small cell lung carcinomas are known to express RET.
- the chimeras can also be used to treat small cell lung carcinomas.
- the eliciting of trophic support and/or differentiation can be achieved by administering a chimera along with a GFR ⁇ l polypeptide (and/or GFR ⁇ 2 or GFR ⁇ 3, for the neurturin or artemin mimics, respectively) or by administering a polynucleotide encoding the chimera and the appropriate GFR ⁇ polynucleotide using the methods previously described with GDNF in Treanor et al . , Na ture 382 : 80-83, 1996. Sequences for GFR ⁇ l are known in the art.
- the invention chimeras are believed to bind to GFR ⁇ l (and GFR ⁇ 2 or GFR ⁇ 3 in the case of the neurturin or artemin mimics, respectively) in the absence of RET and it is believed that the resulting ligand/coreceptor complex is capable of binding to and activating RET receptors expressed by a target cell.
- the chimera and GFR ⁇ polypeptides are from the same species, i.e., human. It is also preferred that the GFR ⁇ polypeptide be in soluble form, i.e., that it lack the GPI linkage to avoid potential undesirable interactions with cell membranes.
- GFR ⁇ polypeptide is intended to include the mature protein with or without the GPI anchor, as well as GFR ⁇ fragments, particularly soluble fragments lacking a GPI anchor, that are capable of binding to both the invention chimeras and RET with such binding leading to the activation of RET.
- compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral .
- Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of a slow release formulation.
- administration can be by injection or infusion into the cerebrospinal fluid (CSF) .
- CSF cerebrospinal fluid
- administration can be with one or more agents capable of promoting penetration of the chimera across the blood- brain barrier.
- the invention chimeras can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
- a chimera can be coupled to any substance known in the art to promote penetration or transport across the blood-brain barrier such as an antibody to the transferrin receptor, and administered by intravenous injection (See for example, Friden et al . , Science 259: 313-311 , 1993).
- a chimera can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties. (See for example Davis et al. Enzyme Eng 4 : 169-73, 1978; Burnham, Am J Hosp Pharm 51 : 210-218, 1994) .
- a chimera of the present invention is administered with a carrier such as liposomes or polymers containing a targeting moiety to limit delivery of the chimera to targeted cells.
- a carrier such as liposomes or polymers containing a targeting moiety to limit delivery of the chimera to targeted cells.
- targeting moieties include but are not limited to antibodies, ligands or receptors to specific cell surface molecules.
- the compositions can also include absorption enhancers which increase the pore size of the mucosal membrane.
- absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl- ⁇ -cyclodextrin, lauroyl- 1-lysophosphatidylcholine and other substances having structural similarities to the phospholipid domains of the mucosal membrane.
- the compositions are usually employed in the form of pharmaceutical preparations . Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used.
- a suitable buffer be present in the composition.
- Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
- the primary solvent can be aqueous or alternatively non-aqueous.
- the invention chimeras can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment .
- the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
- the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood- brain barrier.
- excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or periodic infusion.
- Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used.
- formulations containing a chimera are to be administered orally.
- Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
- suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
- the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
- the compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
- the formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, for example, surface active agents.
- the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
- the dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art . Such calculations can be made without undue experimentation by one skilled in the art based on the activity of the chimera for a particular cell type in vi tro .
- the activity of invention chimeras on various peripheral and central neurons in culture is described below and its activity on a particular target cell type can be determined by routine experimentation. Exact dosages are determined in conjunction with standard dose-response studies.
- the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.
- an invention chimera may be therapeutically administered by implanting into patients vectors or cells capable of producing a biologically-active form of the chimera or a precursor of the chimera, i.e. a molecule that can be readily converted to a biological-active form of the chimera by the body.
- cells that secrete the chimera may be encapsulated into semipermeable membranes for implantation into a patient.
- the cells are transformed to express and secrete both the chimera and the appropriate GFR ⁇ , preferably in a soluble form. It is preferred that the cell be of human origin and that the chimera be derived from human GDNF family ligands when the patient is human.
- the formulations and methods herein can be used for veterinary as well as human applications and the term
- patient as used herein is intended to include human and veterinary patients .
- an invention chimera can be used to promote the ex vivo survival or expansion of cells for transplantation or engraftment.
- Current methods have used bioreactor culture systems containing factors such as erythropoietin, colony stimulating factors, stem cell factor, and interleukins to expand hematopoietic progenitor cells for erythrocytes, monocytes, neutrophils, and lymphocytes (Verfaillie, Stem Cells 12 : 466-476, 1994) .
- stem cells can be isolated from the marrow of human donors, from human peripheral blood, or from umbilical cord blood cells.
- the expanded blood cells are used to treat patients who lack these cells as a result of specific disease conditions or as a result of high dose chemotherapy for treatment of malignancy (George, Stem Cells 12 (Suppl 1) : 249-255, 1994).
- autologous transplants can be performed by removing bone marrow cells before chemotherapy, expanding the cells ex vivo using methods that also function to purge malignant cells, and transplanting the expanded cells back into the patient following chemotherapy (for review see Rummel and Van Zant, J Hema totherapy 3 : 213-218, 1994).
- an invention chimera with or without the appropriate GFR ⁇ can be used for the ex vivo expansion of precursor cells in the nervous system.
- Transplant or engraftment of cells is currently being explored as a therapy for diseases in which certain populations of neurons are lost due to degeneration such as, for example, in Parkinson's disease (Bjorklund, Curr Opin Neurobiol 2 : 683-689, 1992) and spinal cord injury.
- Neuronal precursor cells can be obtained from animal or human donors or from human fetal tissue and then expanded in culture using a chimera of the present invention. These cells can then be engrafted into patients where they would function to replace some of the cells lost due to degeneration.
- neurotrophins have been shown to be capable of stimulating the survival and proliferation of neuronal precursor cells such as, for example, NT-3 stimulation of sympathetic neuroblast cells (Birren et al . , Develop 119 : 597-610, 1993), the chimera could also function in similar ways during the development of the nervous system and could be useful in the ex vivo expansion of neuronal cells.
- Example 1 This example illustrates that the N-terminal extension of GDNF is not required for activity.
- Rat GDNF was the basis for all mutagenesis in this example because its structure has been determined (Eigenbrot and Gerber, Nat. Struct . Biol . 4 : 435-438, 1997).
- amino acid sequence of rat GDNF SEQ ID NO: 12
- mouse neurturin SEQ ID NO: 14
- persephin SEQ ID NO: 14
- the resulting sequence was N-...RRAR ⁇ PGA- HHHHHHDYKDDDDK- RGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCEAAETMYDKILKNLSRSRRLTSDKVGQACCRPVAF DDDLSFLDDSLVYHILRKHSAKRCGCI .
- the mutant proteins were produced in COS cells as follows.
- expression plasmids were transfected using the DEAE-dextran/Chloroquine method (Seed and Aruffo, Proc . Na tl . Acad. Sci . U. S . A . 84 : 3365-3369, 1987).
- COS cells were plated onto 10 cm or 15 cm dishes, transfected, and switched to Delbecco's Modified Eagle's Media containing 1% fetal calf serum. After 4-5 days, conditioned medium was collected, cleared, and either concentrated using Centriprep-10 concentrators (Amicon) or purified by nickel chromatography (Qiagen) .
- Proteins were visualized by immunoblotting with anti-FLAG ® M2 monoclonal antibody (Sigma) . Relative quantities of FLAG ® -tagged proteins were determined by an ELISA also using the anti-FLAG ® M2 antibody. The absolute concentration of GDNF in purified and conditioned medium samples was determined for the G-hf-GDNF construct using the GDNF E- MAX ELISA kit following the manufacturer's instructions (Promega). As shown in Figure 2B, monomeric G-hf-GDNF ran at -25 kD, and N-hf-GDNF at -21.5 kD, consistent with processing at the predicted RXXR cleavage sites from GDNF and neurturin.
- N-hf-GDNF produces GDNF with the 40 amino acid N-terminal extension replaced by two amino acids and the tandem His-FLAG® tags.
- G- hf-GDNF pro-region
- N-hf-GDNF neurturin pro-region
- the Gal4-Elkl/Gal4-luciferase reporter system was used as described previously (Baloh et al., supra ) .
- This system monitors the level of RET activation of the MAP kinase pathway, which induces the Gal4-Elkl fusion protein to activate the Gal4-luciferase reporter, giving a facile measure of receptor activation (Worby et al., J Biol Chem 273 : 3502-3508, 1998; York et al., Na ture 392 : 622-626, 1988).
- RET-3T3 cells were plated at 85,000 cells/well in 12-well plates, and transfected using Superfect (Qiagen) with the reporter plasmids (250 ng/well Gal4-Luc, 50 ng/well Gal4-Elk) , CMV-lacZ (50 ng/well) for transfection normalization, CMV-GFR ⁇ l (250 ng/well) expression plasmid, the mutant GFL construct (250 ng/well) and 650 ng/well pBluescript as carrier for a total of 1.5 ⁇ g DNA/well. Cells were switched to 0.5% serum- containing medium the morning after transfection and harvested 36 hours later.
- the N-terminal extension before the first structural cystine- knot cysteine is highly variable amongst the TGF- ⁇ superfamily members.
- TGF- ⁇ 2 it exists as a short ⁇ -helix and is stabilized by an additional disulfide bond relative to other members of the superfamily (Daopin et al., supra ; Schlunegger and Grutter, Na ture 358 : 430-434, 1992) .
- GDNF and OP-1 the only other members of the TGF- ⁇ superfamily that have been crystallized, the 35-37 residue N-terminal extension was unresolved and therefore does not adopt a consistent conformation in solution (Eigenbrot and Gerber, supra ; Griffith et al., Proc. Natl.
- Example 2 This example illustrates the use of homologue scanning to identify critical GDNF regions for activation of GFR ⁇ l-RET and GFR ⁇ 2- RET. Although- the crystal structure of GDNF is known, the molecular determinants for GDNF-GFR ⁇ receptor binding and specificity are currently undefined.
- Expression plasmids for these mutants were produced as described in Example 1 using fusion PCR, and all were based on the N-hf-GDNF construct. All mutants were produced at similar quantities when transfected into COS cells, and processing of each appeared normal ( Figure 3B) .
- GDNF GDNF.
- Molecular modeling and analysis was done using the SwissPDB viewer (Guex and Peitsch, Electrophoresis 18 : 2714-2723, 1997), and models were rendered using Mole software (Applied Thermodynamics) .
- the GDNF structure was modeled after entry 1AGQ (Eigenbrot and Gerber, supra ) of the Protein Data Bank.
- the 'C/D' dimer from 1AGQ is shown, with the missing segment of monomer ⁇ C built as determined for monomer ⁇ D' .
- the F2a region maps to the bottom of the GDNF molecule along the second finger ( Figure 4B; Eigenbrot and Gerber, supra ) .
- the only other notable mutant that consistently showed activity 60% or lower than full-length GDNF was GPG-F2c, which maps to the strand adjacent to region F2a along the bottom of the molecule .
- GDNF is also capable of activating the GFR ⁇ 2-RET receptor, albeit with a lower affinity than neurturin (Baloh et al., Neuron 18 : 793-802, 1997 Sanicola et al., Proc . Na tl . Acad. Sci . , USA 94 : 6238-6243, 1997; Suvanto et al . , Hum . Molec . Genet . 6: 1267-1273, 1997), whereas persephin cannot (Baloh et al . , 1998, supra ; Milbrandt et al . , 1998, supra) .
- Example 3 This example illustrates that regions F2a and F2c from GDNF, neurturin, or artemin are sufficient for activating GFR ⁇ l-RET.
- the homologue-scanning screen disclosed in Example 2 suggests several regions that are necessary for the full ability of GDNF to activate the GFR ⁇ l-RET receptor.
- using only loss of function analysis it is difficult to assess whether critical receptor-contact residues are being altered directly, or if the mutations induce structural changes that alter receptor contact surfaces elsewhere on the molecule.
- persephin mutants were generated with the putative critical regions from GDNF replacing the corresponding regions of persephin. It was then determined if these "PGP" mutants had gained the ability to activate the GFR ⁇ l-RET receptor complex ( Figure 5) .
- GFR ⁇ l-transfected Neuro2a cells (which endogenously express RET) were then treated with either GDNF or mutant PGP-F2ac as follows.
- Neuro2a cells were transiently transfected using Superfect with an expression plasmid for rat GFR ⁇ l with a FLAG ® tag inserted by PCR mutagenesis at after the signal sequence.
- Cells were treated with 25 ng/mL of the indicated factor for 6 hours, the cells were washed once with cold PBS, lysed, and immunoprecipitated using an anti-FLAG ® M2 monoclonal antibody conjugated to agarose (Sigma) . After washing, the immunoprecipitated samples were visualized by immunoblotting with an anti-RET antibody (C-9; Santa Cruz) .
- Both GDNF and PGP-F2ac induced comparable receptor complex formation, demonstrating that PGP-F2ac had gained the ability to bind and induce GFR ⁇ l-RET complex formation ( Figure 5C) .
- mutant PGP-F2ac was also able to elicit biological responses comparable to GDNF, its ability to induce proliferation in the NBL-S neuroblastoma cell line was determined.
- This cell line expresses GFR ⁇ l and RET and responds to GFR ⁇ l-RET agonists (GDNF, neurturin, artemin) by proliferation.
- the NBL-S proliferation assay was performed as described in Baloh et al., 1998, supra .
- Mutant PGP-F2ac purified from transiently transfected COS cell conditioned medium induced robust proliferation of NBL-S cells, similar to GDNF at all doses, whereas persephin did not (Figure 5D) .
- PGP-F2ac was also able to induce neurite outgrowth in the GFR ⁇ l-RET expressing neuroblastoma line SH-SY5Y (as evaluated according to the method of Baloh et al . , 1998, supra ) similar to GDNF ( Figure 5E) .
- Neurturin and artemin are also able to activate GFR ⁇ l-RET (Baloh et al., 1998, supra ; Creedon et al . , Proc . Na tl . Acad. Sci . US 94 : 7018-7023, 1997), and therefore it was determined if the same regions (F2a and F2c) from these two additional GFR ⁇ l agonists are also sufficient to activate the GFR ⁇ l-RET receptor ( Figure 6) .
- homologue-scanning mutagenesis is ideally suited for identifying sites involved in differential receptor specificity, it cannot delineate all residues involved in receptor binding, as some of these are identical in GDNF and persephin.
- residues from region F2a and F2c are sufficient when placed in the context of persephin to activate GFR ⁇ l-RET indicates that residues in these regions are likely to be directly involved in binding to GFR ⁇ 's.
- residues from region Ha lie directly adjacent to regions F2a and F2c from the finger 2 region of the opposing monomer, and form continuous surfaces along the side and bottom of the molecule (see Figure 4C) . Because these regions are all critical for GFR ⁇ specificity, it appears that the molecular surface formed by the heel of one monomer and the second finger of the adjacent monomer form GFR ⁇ binding surfaces on GDNF.
- VEGF vascular endothelial growth factor
- VEGF vascular endothelial growth factor
- regions F2a and F2c regions F2a and F2c identified here for GDNF.
- the receptor binding site on VEGF involves residues from finger 2 and the analogous heel region of the opposing monomer.
- mutagenesis of TGF- ⁇ indicates that the binding affinity of the different TGF- ⁇ isoforms for the TGF- ⁇ type II receptor is also determined by residues along the second finger of the molecule, analogous in location to region F2c
- Example 4 This example illustrates that additional determinants are critical for neurturin to activate GFR ⁇ 2-RET and artemin to activate GFR ⁇ 3-RET.
- mutants PGP-F2ac and PNP-F2ac cannot activate GFR ⁇ 2-RET s consistent with the homologue-scanning mutagenesis in Example 2 ( Figure 4), which suggested that regions in addition to F2a and F2c are required for GDNF to activate GFR ⁇ 2-RET.
- mutant PGP-Ha was significantly attenuated n its ability to activate GFR ⁇ 2-RET (-27%) , whereas it showed a minor loss in the ability to activate GFR ⁇ l-RET (-80%; Figure 4).
- region Ha contains additional molecular determinants required for activating GFR ⁇ 2-RET
- a persephin mutant was generated with regions F2a, F2c and Ha replaced by the corresponding regions from neurturin, the highest affinity GFR ⁇ 2 agonist (PNP-Ha/F2ac; Figure 7A) . While mutant PNP-F2ac elicited only a minor increase in GFR ⁇ 2- RET activation as before, mutant PNP-Ha/F2ac was significantly better at activating GFR ⁇ 2-RET, however it did not restore full activity (usually 70-80% of GDNF; Figure 7B) .
- mutant PAP-F2ac was entirely inactive, mutant PAP-Ha/F2ac regained significant activity (ca. 70-80% of full length artemin; Figure 7D) . Therefore, similar to the neurturin-GFR ⁇ 2 interaction, the artemin-GFR ⁇ 3 interaction requires molecular determinants from both the heel and finger 2 regions of the molecule, whereas interaction of GDNF, neurturin or artemin with GFR ⁇ l only requires regions F2a and F2c from finger 2.
- GFR ⁇ l-RET is the most promiscuous of the GFL receptors, able to interact with three of the four known ligands. This is consistent with GFR ⁇ l-RET having the most minimal requirements for being activated (regions F2a and F2c) , and additional regions being required for activating GFR ⁇ 2-RET and GFR ⁇ 3-RET.
- a cell survival assay using rat cerebellar granule cells in culture was developed. These cells do not express endogenous RET or GFR ⁇ coreceptors and can be transfected efficiently, therefore providing a model system of cell survival with defined receptor components.
- Rat cerebellar granule cell dissection and culture was performed as described previously Miller and Johnson, J. Neurosci . 1 6: 7487-7495, 1996.
- Timed-pregnant Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) .
- cerebella were dissected, cut into 1 mm pieces, and incubated for 15 min in 0.3 g/ml trypsin (Worthington, Freehold, NJ) at 37°C. The pieces were triturated with a fire-polished Pasteur pipette in the presence of trypsin and the resulting cell suspension was passed through a Nitex filter (size 3-.20/14; Tetko, Elmsford, NY). Cells were plated at a density of 2.3xl0 5 cells/cm 2 in four-well dishes (Nunc) coated with 0.1 mg/ml poly-L-lysine .
- Plating medium (K25 +S) consisted of Basal Medium Eagle (Life Tech, Gaithersburg, MD) containing 10% dialyzed fetal bovine serum, 20 mM KC1, 100 U/ml penicillin and 100 ug/ml streptomycin. To reduce the number of non- neuronal cells, 3.3 ⁇ g/ml aphidocholine was added to the medium 24 hrs after plating.
- Granule cell transfections were performed using a modified calcium phosphate protocol as described previously (Moulder et al . , J. Neurosci . 19 : 705-715, 1999). Expression plasmids for GFR ⁇ l, GFR ⁇ 2 and RET are described elsewhere (Baloh et al . , 1997, supra ) . At 5 days in vitro (DIV) , medium was replaced with Dulbecco's Modified Eagle's Medium (Life Tech) for 1 hr.
- DIV Dulbecco's Modified Eagle's Medium
- the number of initial EGFP- positive cells in a defined area (minimum of 150 cells) of two to four wells (of a four-well dish) per condition were counted 24 hrs after transfection, then rinsed the cultures twice in Dulbecco's Modified Eagle's Medium and switched them to high-potassium/serum (K25+S) ; low potassium/no serum (K5-S) medium alone; or K5-S supplemented with the indicated factors for 48 hrs. After this period, the number of EGFP-positive cells remaining in the originally defined area of the well was counted again to obtain the percentage neuronal survival.
- Cerebellar granule cells transfected with RET or GFR ⁇ l alone do not survive in the absence of high potassium or in the presence of GDNF, however cells cotransfected with GFR ⁇ l and RET survive in the presence of GDNF at levels comparable to the control condition of high potassium plus serum ( Figure 8A; Miller and Johnson, supra). Consistent with other assays, cerebellar granule cells transfected with GFR ⁇ l-RET or GFR ⁇ 2-RET survive in the presence of either GDNF or neurturin, but not persephin ( Figure 8B) . However, mutant PGP-F2ac was only capable of supporting the survival of GFR ⁇ l-RET transfected, but not GFR ⁇ 2-RET transfected cells.
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See also references of WO0147946A2 * |
Also Published As
Publication number | Publication date |
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US6866851B1 (en) | 2005-03-15 |
CA2396039A1 (en) | 2001-07-05 |
EP1299410A4 (en) | 2004-06-23 |
WO2001047946A9 (en) | 2002-05-23 |
JP2004515205A (en) | 2004-05-27 |
WO2001047946A2 (en) | 2001-07-05 |
AU3265501A (en) | 2001-07-09 |
WO2001047946A3 (en) | 2003-02-06 |
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